Volume 19, Issue 3, Pages 372-380 (March 2012) EccA1, a Component of the Mycobacterium marinum ESX-1 Protein Virulence Factor Secretion Pathway, Regulates Mycolic Acid Lipid Synthesis  Shilpa A. Joshi, David A. Ball, Mei G. Sun, Fredric Carlsson, Brigitte Y. Watkins, Nina Aggarwal, Jenna M. McCracken, Kassidy K. Huynh, Eric J. Brown  Chemistry & Biology  Volume 19, Issue 3, Pages 372-380 (March 2012) DOI: 10.1016/j.chembiol.2012.01.008 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 EccA1's ATPase Activity Is Needed for ESAT-6 Secretion and Hemolytic Activity (A) EccA1 contains an N-terminal tetratricopeptide repeat (TPR) domain and a C-terminal ATPase domain. (B) Wild-type and plasmid-encoded wild-type or EQ EccA1 are expressed at similar levels. CF, culture filtrate; CL, whole-cell lysate; GroEL, a loading control. (C) EQ-EccA1 expression cannot restore ESAT-6 secretion. Ag85 complex is a loading control, and 8-fold more normalized CF than CL was analyzed in the Ag85 blot, whereas a 1:1 CF:CL ratio was analyzed in all other blots. (D) Wild-type EccA1 complements eccA1::Tn's hemolytic defect, but EQ-EccA1 expression is similar to the buffer control. Averaged data are from three independent experiments and are mean values ± SEM. See also Figure S1. Chemistry & Biology 2012 19, 372-380DOI: (10.1016/j.chembiol.2012.01.008) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 EQ EccA1 Interacts More Stably with Lipid Synthases (A) Cytosolic complexes containing EccA1 were isolated and visualized by SDS-PAGE and silver staining. EQ EccA1 coprecipitates more proteins larger than 100 kDa compared to wild-type. (B) Mass spectrometric analyses of coprecipitating proteins identified multiple polyketide synthases as interacting better with EQ EccA1. (C) Immunoblotting of coprecipitation reactions with specific antibodies shows more Pks13 and Mas interacting with EQ EccA1 (left). Quantitated values from two independent experiments are shown (right). (D) All EccA1 variants interact with Pks131–1340, KasB, and KasA as strongly as a control in a two-hybrid experiment, and less well with MmaA4. Percent interaction is calculated from the ratio of colonies on selective plates to those on nonselective plates. Averaged data are from two or more independent experiments, and mean values ± SEM are shown. See also Figure S2 and Tables S1 and S2. Chemistry & Biology 2012 19, 372-380DOI: (10.1016/j.chembiol.2012.01.008) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 EccA1 and the FAS-II Mycolic Acid Synthesis System Interact Functionally (A) Radiolabeled and isolated mycolic acid methyl esters were resolved. (B) Quantitation revealed mycolic acid synthesis defects in eccA1::Tn and (eccA1::Tn)/EQ. Averaged data are from three or more independent experiments, and mean values ± SEM are shown (∗∗p ≤ 0.03). Chemistry & Biology 2012 19, 372-380DOI: (10.1016/j.chembiol.2012.01.008) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 EQ-EccA1 Expression Also Sensitizes Cells to Ethionamide Stress (A) Left: ethionamide inhibition of mycolic acid synthesis is plotted. (eccA1::Tn)/EQ is most inhibited. Averaged data are from at least four independent experiments, and mean values ± SEM are shown (∗p < 0.05; ∗∗∗p ≤ 0.01). Right: IC50 values for growth-inhibition sensitivity to ethionamide are shown. (eccA1::Tn)/EQ is uniquely sensitized to ethionamide. Mean values ± SEM are averaged data from five independent experiments (∗∗∗p ≤ 0.01; ∗∗∗∗p ≤ 0.002). (B) IC50 values for growth-inhibition sensitivity to two hydrophobic antibiotics, rifampin and erythromycin, are shown. (eccA1::Tn)/EQ is similar to eccA1::Tn in being more resistant to these antibiotics. Mean values ± SEM are averaged data from four or more independent experiments (∗p < 0.05; ∗∗∗p ≤ 0.01). Chemistry & Biology 2012 19, 372-380DOI: (10.1016/j.chembiol.2012.01.008) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 EQ EccA1 Further Decreases Virulence and Intracellular Growth (A) An EQ-expressing integrant strain is attenuated for zebrafish killing (∗∗∗∗p ≤ 0.002 compared to wild-type, log-rank test; n ≥ 5 for each strain). (B) All strains grew similarly in 7H9 media. (C) eccA1::Tn is defective for intracellular growth in bone marrow-derived macrophages. Wild-type EccA1 complements this defect, but EQ EccA1 worsens growth. Data are averaged from three independent experiments (∗∗p ≤ 0.03; ∗∗∗p ≤ 0.01; ∗∗∗∗p ≤ 0.002). Chemistry & Biology 2012 19, 372-380DOI: (10.1016/j.chembiol.2012.01.008) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 6 A Model of EccA1's Interaction with FAS-II Proteins For efficient mycolic acid production, EccA1 must interact with FAS-II components such as Pks13, KasB, and KasA (A–C), and release them in an ATP hydrolysis-linked manner to the rest of FAS-II (D). (eccA1::Tn)/EQ is further sensitized to mycolic acid stress, relative to eccA1::Tn, because it has a lower amount of fully functioning Pks13, KasB, and KasA, important in (D). EccA1's interactions with these partner proteins are shown to be direct and exclusive here, but other mycobacterial proteins may aid these interactions or be part of these complexes. Three ATPs (shown in red) are bound to hexameric EccA1 here, but the real number is unknown. Chemistry & Biology 2012 19, 372-380DOI: (10.1016/j.chembiol.2012.01.008) Copyright © 2012 Elsevier Ltd Terms and Conditions